Overcoming of BRAF inhibitor resistance in melanoma

XY Chan, K Brassington, IA Darby and TJ Piva

School of Health & Biomedical Sciences, RMIT University, Bundoora, Vic 3083, Australia

Vemurafenib (PLX4032) is often used to treat melanomas that possess the BRAFV600E mutation. While the tumours are shown initially to regress, they become resistant to the drug and the patient relapsed and eventually dies. It has been shown that the growth factors secreted by adjacent cells activated signalling pathways in these melanoma cells. Using specific signalling pathway inhibitors, we investigated the growth factor-activation of intracellular signalling pathways in two melanoma (MM418-C1 and D24) cell lines. MM418-C1 melanoma cells possess the BRAFV600E mutation, while D24 melanoma cells do not. Growth factors (HGF or TGFα) were added to cells primed for 24 h in low serum (0.5% FBS), and the expression of p-BRAF, p-ERK1/2, p-p38, p-JNK1/2 as well as p-Akt were quantified using Western blots. We discovered that HGF signalled through BRAFV600E in MM418-C1 cells, but TGFα signalled through BRAFWT in D24 cells. However, expression of other downstream signalling intermediates (p-ERK1/2, p-p38 and p-JNK1/2) of BRAF was increased when either HGF or TGFα was added in both cell lines. The effects of growth factors and/or specific signalling pathway inhibitors on cell migration scratch assay were also examined. The results obtained suggest the involvement of alternative signalling pathways and the PI3K-AKT-mTOR pathway was also investigated in this study. The significance of these findings will be discussed.